Summary Natural Killer T (iNKT) cells represent a lymphocyte population that has evolved to recognize glycolipid antigens presented by CD1d. Upon recognition of glycolipids, iNKT cells respond within hours, reminiscent of innate rather than adaptive functions. These cells have been implicated in the regulation of immune responses associated with a broad range of diseases, including autoimmunity, infectious diseases and cancer. iNKT cells express a highly restricted TCR repertoire, due to the usage of an invariant V[unreadable]14-J[unreadable]18 sequence and the preferential usage of the V[unreadable]8.2, V[unreadable]7 and V[unreadable]2 gene segments. The most diversity of the TCR structure is limited to the CDR3 region of the TCR[unreadable] chain. Our recent results suggest that the role of the TCR[unreadable] chain is to modulate the overall affinity of the TCR for the antigen/CD1d complex rather than to facilitate recognition of different antigens. The overall goal of this grant is to understand more fully the formation of the iNKT cell repertoire and whether high affinity iNKT-cell-derived TCRs can be used as functional tools in the fight against cancer. The experiments proposed will examine: 1) whether the V[unreadable] bias of the iNKT cell repertoire is dictated by the ability of each V[unreadable] to bind CD1d, 2) whether iNKT cell precursors that express high affinity TCRs are negatively selected during development and 3) whether high affinity iNKT cell- derived TCRs can be used for adoptive cell transfer therapy against cancer. A better understanding of glycolipid recognition by the iNKT TCR and how it might affect iNKT cell functions will allow for the rational optimization of iNKT cell ligands and will define guidelines for fine tuning iNKT cell function.